2-DE was performed horizontally using an IPGphor and Multiphor (GE Healthcare) setup according to a method previously described by Görg, et al. [19 (link)], used by our group in the analysis of macrophage cell samples [18 (link)]. Macrophage protein samples, approximately 150 μg of protein per sample, were separated by 2-DE, with the following experimental replicates: 5 untreated control; 5 2mix treated; 3 7-ketocholesterol treated; 3 cholesterol treated; and 3 ethanol treated. First dimension isoelectric focussing was performed on pH 3–10 non-linear IPG strips (GE Healthcare) for 46000 Vh. Second dimension was performed using home cast homogenous gels (SDS-PAGE; T = 14%, C = 1.5%) and run overnight. 2-DE gels were stained using silver nitrate, according to the protocol set by Shevchenko, et al. [20 (link)]. Stained gels were imaged using a charge-coupled device camera and VersaDoc 4000MP system (Bio-Rad Laboratories, CA, USA) and gels spots matched and quantified, as parts per million (ppm) of total gel density, between gels using PD-Quest v8.0.1 (Bio-Rad Laboratories). Statistical analysis of matched protein spots between control untreated cells and treatments; 2mix, 7-ketocholesterol, cholesterol and ethanol was performed using a non-parametric Mann-Whitney U test (SPSS v21; IBM, UK), where significance was set at p ≤ 0.05.
Versadoc 4000mp system
Manufactured by Bio-Rad
Sourced in United States, China
The VersaDoc 4000MP system is a multifunction imaging system designed for versatile imaging applications. It features a high-resolution CCD camera, adjustable zoom lens, and variable illumination sources to capture images of a variety of samples, such as gels, blots, and chemiluminescent and fluorescent samples. The system is capable of acquiring and processing high-quality images for various analytical purposes.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (13 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Bca protein assay kit, by Beyotime (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Bs1699, by Bioworld Technology (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Ap0060, by Bioworld Technology (3 mentions)
Bs7319, by Bioworld Technology (2 mentions)
Ap0707, by Bioworld Technology (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Bs6625, by Bioworld Technology (2 mentions)
Ab79847, by Abcam (2 mentions)
Bs2192, by Bioworld Technology (2 mentions)
Ecl chemiluminescence detection kit, by Vazyme (2 mentions)
Ecl chemiluminescence detection kit, by Thermo Fisher Scientific (2 mentions)
Anti c ebpα, by ZenBio (2 mentions)
Anti foxo1, by ZenBio (2 mentions)
Anti adipoq, by ZenBio (2 mentions)
Ripa lysis buffer, by Vazyme (2 mentions)
Pd quest v8, by Bio-Rad (1 mentions)
26 protocols using versadoc 4000mp system
1
Two-Dimensional Gel Electrophoresis for Macrophage Proteomics
2
Quantifying Gag-containing Complexes by Sucrose Gradient
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Expression of Gag-containing complexes was assayed as previously described (51 (link), 53 (link)) with a few modifications. Gag-expressing cells were lysed in lysis buffer A (10 mM HEPES [pH 7.5], 10 mM NaCl, 1 mM magnesium acetate, 0.85% octyl-β-d- glucopyranoside) for 10 min on ice, and the lysates were spun at 500 × g for 5 min to clear insoluble nuclei. The lysates were then loaded onto the top of an 11-ml 10% to 60% sucrose gradient and spun in the ultracentrifuge using a Beckman Coulter SW41Ti rotor at 35,000 rpm for 3 h. Centrifuged samples were separated into 300-µl aliquots. A 100-µl volume of each fraction was added to 200 µl distilled water (dH2O), and all fractions were subsequently blotted onto low-fluorescence polyvinylidene difluoride (PVDF) membranes (Bio-Rad) using a microsample filtration manifold (Schleider & Schuell). On a separate membrane, serial dilutions of the VLP-containing supernatant collected as described above were also loaded onto PVDF membranes. All membranes were then subjected to standard Western blotting using a fluorescently labeled α-P24 antibody (Millipore MAB880-AF). Gag signal was detected using a Bio-Rad Versadoc 4000MP system and quantified using Image Studio 5.2.
3
Liver Protein Extraction and Western Blotting
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Total protein extraction and western blotting were performed as previously described (Zhao et al., 2021 ). Following the instructions, the protein concentration was measured using the BCA Protein Assay kit (Rockford, IL). Thirty micrograms of total liver protein were subjected to electrophoresis on the 10% SDS-PAGE gel and transferred to the nitrocellulose membrane for subsequent immunoblotting. Western blotting analysis for DNMT1 (Abclonal, A19679) and tubulin-α (Bioworld, BS1699) were carried out according to the manufacturer's protocols. The immunoblot images were captured using VersaDoc 4000MP system (Bio-Rad). The band density was analyzed by Quantity One software (Bio-Rad).
4
Liver Protein Extraction and Western Blot Analysis
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Total protein was extracted from 80 mg frozen liver sample as previously described51 (link). The protein concentration was determined according to the manufacturer’s instructions of Pierce BCA Protein Assay kit (Rockford, IL, USA). Forty or sixty micrograms of protein were used for electrophoresis on a 7.5% or 10% SDS-PAGE gel. Western blot analysis for methionine adenosyltransferase 2B (MAT2B) (15952–1-AP, Proteintech, USA, diluted 1:1000), DNA (cytosine-5-)-methyltransferase 1 (DNMT1) (sc-20701, Santa Cruz, UK, diluted 1:200), GR (sc-1004, Santa Cruz, UK, diluted 1:200), 11β-HSD type 1 (11β-HSD1) (ab109554, Abcam, UK, diluted 1:1000), Type 2 11β-HSD (11β-HSD2) (sc-20176, Santa Cruz, UK, diluted 1:200), PPARα (ab2779, Abcam, UK, diluted 1:1000), mitochondrial transcription factor A (TFAM) (BS7319, Bioworld Technology, USA, diluted 1:1000), COX4 (AP0707, Bioworld Technology, USA, diluted 1:1000) were carried out according to the recommended protocols provided by the manufacturers. Images were captured by VersaDoc 4000MP system (Bio-Rad, USA) and the band density was analyzed with Quantity One software (Bio-Rad, USA).
5
Western Blot Analysis of GRα
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Total protein extracts with RIPA lysis buffer (Beyotime Biotechnology, Jiangsu, China) and protein quantities were measured by the BCA Protein Assay kit (Beyotime Biotechnology, Jiangsu, China). Antibodies against GRα (sc-393232, Santa Cruz, USA) and tubulin (sc-5274, Santa Cruz, USA) was used in Western blot analysis. Images were captured with VersaDoc 4000MP system (Bio-Rad).
6
Adrenal Protein Extraction and Quantification
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Total protein was extracted from 15 mg frozen adrenal samples, as previously described [24 ]. BCA Protein Assay kit (No. 23225, Thermo Scientific) was used to measure protein concentrations following manufacturer’s instructions. Fifty micrograms of protein were used for electrophoresis on a 10% SDS-PAGE gel. Western blot analysis was performed following the protocols provided by the manufacturers. Tublin-β (AP0064, Bioworld, USA, diluted 1:5,000) was used as an internal reference. VersaDoc 4000MP system (Bio-Rad, USA) was employed to capture the images and Quantity One software (Bio-Rad, USA) was used to analyze the band density.
7
Protein Extraction and Western Blot Analysis
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For protein extraction, 1 hundred oocytes or their companion CCs were pooled as one sample (four samples per group) and lysed in Laemmli sample buffer (SDS sample buffer with 2-mercaptoethanol), and boiled at 100 °C for 5 min. Approximately 15 μg of total protein from each sample were subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE). Mouse anti-β-actin antibody (AP0060, Bioworld, 1:200) and mouse anti-γ-actin antibody (ab123034, Abcam, 1:200) were used as primary antibodies. Western blot analyses were carried out according to the recommended protocols provided by the manufacturers. Coomassie brilliant blue staining method was used to calibrate the target proteins expression levels, as reported previously [26 (link)]. Images were captured by VersaDoc 4000MP system (Bio-Rad, Hercules, CA) and the band density was analyzed with Quantity One software (Bio-Rad).
8
Protein Quantification and Western Blot Analysis
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The protein concentrations were measured by BCA Protein Assay kit (No.23225; Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. A total of 40-μg proteins were used for electrophoresis on a 10% and 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred onto a nitrocellulose membrane. The primary antibodies used for Western blot analysis are listed in Table 2 . Tubulin-α and β-actin were selected as internal controls. Images were captured by VersaDoc 4000MP system (Bio-Rad, Hercules, CA) and the band density was analyzed with Quantity One software (Bio-Rad).
The Primary Antibodies Used for Western Blot Analysis
| Primary Antibody | Company | Catalog No. | Dilution |
|---|---|---|---|
| METTL3 | Abcam | AB98009 | 1:1000 |
| METTL14 | Abcam | AB98116 | 1:1000 |
| FTO | Abcam | AB77547 | 1:1000 |
| Collagen I | Boster Biological Technology | BA0325 | 1:500 |
| TGF-β1 | Proteintech | 21898-1-AP | 1:1000 |
| α-SMA | Zen bioscience | 252157 | 1:500 |
| IKKγ | Bioworld | BS6772 | 1:1000 |
| p-IKKα/β | Bioworld | BS5083 | 1:1000 |
| p-p65 | Cell Signaling Technology | 3033 | 1:1000 |
| Tubulin-α | Bioworld | BS1699 | 1:5000 |
| β-actin | Bioworld | BS6007M | 1:5000 |
α-SMA, α-smooth muscle actin; FTO, fat mass and obesity–associated protein; p-p65, phosphorylated p65; TGF-β1, transforming growth factor β1.
9
Western Blot Analysis of Protein Expression
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The pancreatic tissue and RIN-m5f cells were lyzed in NP-40 lysis buffer and centrifuged for 20 min (12,000 g, 4 °C). The concentration of protein was then quantitated, and the protein lysate (20 μg) was analyzed using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method, after which it was electrotransferred onto PVDF membranes (Millipore, Billerica, MA). The membranes were blocked by 5% skimmed milk powder and incubated with anti-SIRT1 (81 kDa; 1:1000) (Abcam, Waltham, MA), anti-FoxO1 (69 kDa; 1:1000) (Abcam), anti-LC3 (15 kDa; 1:2000), anti-p62 (62 kDa; 1:1000) (CST) and anti-GAPDH (36 kDa; 1:2000) (Abcam) at 4 °C overnight, and then with the secondary antibodies of horseradish peroxidase (HRP)-conjugated (rabbit) for 2 h at room temperature. Finally, the samples were visualized by enhanced chemiluminescence (ECL) (Millipore, Billerica, MA) on a VersaDoc 4000 MP system (BIO-RAD). The ImageJ software (National Institutes of Health, https://imagej.nih.gov/ij/ ) was used to perform densitometric analysis and calculate the relative protein expression level by comparing with the control (Bethesda, MD).
10
Protein Expression Analysis by Western Blot
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The total protein extracts with the RIPA (Radio immunoprecipitation assay) lysis buffer and protein quantities were measured by the BCA Protein Assay kit (Auragene Bioscience, Changsha, China). Antibodies against PDK1 (NB100, Novus, Littleton, CO, USA), p-PDK1 (9634S, CST, Boston, MA, USA), Akt (9272S, CST, Boston, MA, USA), p-Akt (104A282, Novus, Littleton, CO, USA), IkBα (ABIN926864, 4A Biotech, Beijing, China), p-IkBα (2859, CST, Boston, MA, USA), and β-actin (ab6276, Abcam, Cambridge, MA, USA) were used in the western blot analysis. Images were captured with the VersaDoc 4000MP system (Bio-Rad).
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